La investigación presentada aquí fue apoyado por Charles River. Los autores están muy agradecidos a los modelos obtenidos mediante ingeniería genética y el grupo de servicios y el personal de Embriología de cuidado de los animales y la inyección de la hormona.

1. Criopreservación de espermatozoides de ratón <ol><li> Para cada línea para ser criopreservados, prepare lo siguiente antes de comenzar. <ol><li> Un pozo de una placa de cuatro pocillos con 240 l de kit de CPA </li><li> 15 pajuelas de criopreservación de espermatozoides de la siguiente manera <ol><li> Conecte el pajuelas de 0,25 ml a las jeringas de 1 ml con tapón de algodón cercano a la jeringa. En cada uno de paja, carga de 8 cm (aproximadamente 160 l) de medio de fertilización in vitro, y luego de 1 cm de aire. Etiqueta de la paja con el nombre de la línea para ser preservado. </li></ol></li></ol></li><li> LN 2 debe ser de 15 cm de profundidad en el cuadro de espuma. Cierre la tapa. </li><li> La eutanasia a dos ratones machos de cada línea para ser criopreservados. Los ratones deben ser entre 10 y 60 semanas. Retire los dos epidídimos caudal, junto con el conducto deferente de cada ratón. </li><li> Coloque el epidídimo en el CPA previamente preparado que contiene los pocillos de una placa de 4 pocillos (paso 1.1). </li><li> Hacer 5-7 cortes longitudinales en cada epidídimo y suavementeApriete cada conducto deferente para empujar el esperma hacia fuera. </li><li> Agite la mezcla de CPA y el epidídimo con la mano suavemente a temperatura ambiente durante 3-5 minutos. Esto permitirá a los espermatozoides a nadar. </li><li> Utilizando la paja previamente preparado, aspirar 5 mm (aproximadamente 10 l) de suspensión de esperma y luego de 1 cm de aire. </li><li> Repita este proceso con hasta un total de 15 pajillas. De carga y el sello paja a los 10 minutos para la supervivencia mejores espermatozoides. </li><li> Termosellado ambos lados de la paja con cuidado. Coloque la paja en el recipiente de congelación con la columna de los espermatozoides en primer lugar. </li><li> Coloque la pipeta de 1 ml proporciona como parte del kit de la lata para extender el mango. Coloque el recipiente en LN 2 en el cuadro de espuma a través del agujero en la tapa. Deje que el frasco para flotar verticalmente en la LN 2 durante 10 minutos. </li><li> Sumergir el recipiente en el LN2. Los espermatozoides criopreservados y ahora se puede mover a largo plazo de almacenamiento de LN 2. </li><li> El envase no debe ser utilizado parala siguiente línea hasta que se calienta hasta la temperatura ambiente. </li></ol> 2. Ratón superovulación <ol><li> En general, el uso de ratones hembra jóvenes el mismo fondo que los donantes de esperma. </li><li> Los ratones deben ser ~ 12 g (3-4 semanas de edad). </li><li> Realizar una inyección intraperitoneal con IU 5.10 de embarazadas gonadotropina de suero de yegua (PMSG). </li><li> Espere 46 a 48 horas. </li><li> Realizar una inyección intraperitoneal con IU 5.10 de la gonadotropina coriónica humana (hCG). </li><li> Los ovocitos de la cosecha 14-16 h después de la inyección de hCG como se describe a continuación. </li></ol> 3. Ratón de recuperación de espermatozoides y fecundación in vitro <ol><li> Antes de la FIV, para cada uno de paja para ser descongelado, preparar la siguiente y equilibrar durante al menos una hora en un 37 ° C incubadora de CO 2. <ol><li> Espermatozoides plato de incubación: una placa de Petri de 35 mm, con una caída de 90 l de medio de tratamiento de esperma (STM) cubierto con aceite. </li><li> Corte de plato: Para la recogida de ovocitos de un máximo de 10 superovulated mujeres, una de 35 mm placa de Petri con 3 ml de medio de fertilización in vitro. </li><li> Fecundación in vitro, así: Para cada grupo de 5 mujeres superovuladas para la FIV, un pocillo de una placa de 4 y con 500 l de medio de FIV (si está utilizando 10 hembras superovuladas, por ejemplo, se necesitan 2 pozos...) </li><li> Lavar los platos: una placa de Petri de 35 mm con 3 ml de potasio simple medio optimizado (KSOM) para el lavado de cada pocillo de la placa de FIV 4-bien utilizado </li><li> Placa de cultivo: una placa de Petri de 35 mm con 50 gotas l de medio KSOM cubiertos con aceite para el cultivo de embriones de cada pocillo de la placa de la FIV. </li></ol></li><li> Eliminar una paja de LN 2 y colóquelo en un baño de agua 37 º C durante 2-3 minutos. </li><li> Quitar la paja del baño de agua y limpiar para eliminar el exceso de agua. Haga el primer corte cerca de la primera columna de aire más cercano al tapón de algodón. Cortar la paja en el medio de FIV seguirá habiendo una columna de aire visible antes de la columna de los espermatozoides. </li><li> A continuación, corte el extremo de la paja en el segunda columna de aire, teniendo cuidado de evitar el corte de la columna de los espermatozoides. Corte en un ángulo de 45 - esto hará que sea mucho más fácil para aspirar a los espermatozoides. </li><li> Aspirar la suspensión de esperma desde el extremo distal de la paja con una pipeta 200 microlitros. </li><li> Lugar la suspensión de esperma en el centro de la caída de STM y se incuba la placa durante 45-60 minutos en un 37 ° C al 5% de CO 2 incubadora. </li><li> Durante la incubación de espermatozoides, diseccionar los oviductos de las hembras superovuladas en el plato de corte. Use una aguja de 30 g o fórceps para rasgar cada oviducto, la liberación de los complejos cumulus-ovocito (COC). Evite la grasa y la sangre en el medio de la FIV como sucio para los medios de comunicación hace que el lavado de medidas adicionales. </li><li> Una vez que todos los oviductos se han roto, la transferencia de los anticonceptivos orales combinados de cinco mujeres de cada pozos de fecundación in vitro. El número de AOC producidos por superovulación es muy dependiente de la cepa. </li><li> Recoger 10-15 l de esperma de la periferia de la caída de STM y abonar una fecundación in vitro y aproximadamente 14-16 horas después de la hCG injexión. Repita la fertilización para cada bien utilizado. La concentración de esperma final será de aproximadamente 1 millón / ml. </li><li> Cocultivo de espermatozoides y ovocitos durante 4-6 horas en un 37 ° C 5% de CO 2 incubadora. </li><li> Lavado de los embriones con KSOM un mínimo de 2 veces y la cultura en forma de gotas KSOM para el desarrollo en un 37 ° C 5% de CO 2 incubadora. </li><li> Tasa de fecundación in vitro se ha calculado como dos embriones de células etapa de total de ovocitos utilizados después de la cultura durante la noche. </li></ol> 4. Resultados representante El esperma de 5 cepas puras ratón Charles River se congeló. Las relaciones de protección se calcula dividiendo los valores de evaluación de los espermatozoides congelados en fresco valores de esperma de evaluación. Relaciones de protección de la motilidad de los espermatozoides fueron 60,2%, 81,3%, 78,6%, 66,5%, 61,0% para C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl y BALB / cAnNCrl, respectivamente. Relaciones de protección para la motilidad progresiva rápida fueron 47,3%, 69,4%, 57,0%, 52,8%, 54,1% en C57BL/6NCrl, 129S2/SvPas<html> Crl, FVB / NCrl, DBA/2NCrl y BALB / cAnNCrl, respectivamente (Fig. 1). Las tasas de fecundación in vitro con esperma congelado-descongelado fueron el 55,7%, 26,4%, 87,3%, 87,8% y 26,2% en C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl y BALB / cAnNCrl, respectivamente (Fig. 2) . Durante un período de 1,5 años, 49 líneas de ratones transgénicos fueron archivados por la crioconservación de esperma. Estas líneas fueron más tarde recuperados con éxito (según la definición de los cachorros nacidos vivos) por fecundación in vitro en 4 cepas de mujeres (C57BL / 6, BALB / c, DBA / 1 y 129Sv) (Fig. 3). <img alt="Figura 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> Figura 1. Las relaciones de protección de la motilidad espermática y la motilidad progresiva rápida en los ratones <img alt="Figura 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> Figura 2. Fertilización in vitro con semen congelado-descongelado en ratones 3713fig3.jpg &quot;/&gt; Figura 3. Fecundación in vitro con espermatozoides congelados-descongelados en ratones modificados genéticamente

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

Los autores son empleados de Charles River, el fabricante de este equipo.

Desde 1990, el Protocolo de congelar el semen básico con 18% de rafinosa y 3% de leche desnatada se ha demostrado su fiabilidad en muchas cepas de ratones y un gran número de líneas de ratón han sido criopreservados 1,2,3,4,5,6,7. Las principales causas de daño a las células de esperma durante el proceso de congelación y descongelación se han asociado con la formación de hielo 8,9 y especies reactivas de oxígeno 10. La suplementación con atrapadores de radicales libres, tales como los aminoácidos, y agentes reductores, como monotioglicerol, en el medio de congelación fue investigado y probado para incrementar sustancialmente la tasa de FIV con espermatozoides congelados-descongelados en las cepas puras como los C57BL / 6 11,6. En el presente Protocolo, se desarrolló un nuevo I • Cryo kit para la criopreservación de espermatozoides de ratón mediante la optimización de la CPA básicos disponibles en el mercado con los antioxidantes y los bloqueadores de hielo y modificando la congelación de espermatozoides y la fecundación in vitro. Relaciones de protección de la motilidad de los espermatozoides(&gt; 60%) y la motilidad progresiva rápida (&gt; 45%) se observó que los espermatozoides de cinco cepas de ratones endogámicos congelados con el I • Kit de Cryo. El 2 de células embrión en su fase de desarrollo con tasa de espermatozoides congelados-descongelados en cinco cepas de ratones endogámicos se mejoró notablemente en comparación con el que, con base CPA 11,6. En el uso del kit, el usuario crea una suspensión de esperma concentrado por congelar el semen de dos machos por cada línea. Sólo por congelación 15 pajillas el proceso es rápido, sencillo y ocupa menos espacio de almacenamiento para el almacenamiento de corto o largo plazo. Mayoría de las veces, las líneas de ratón pueden ser recuperados por la descongelación sólo 1 o 2 popotes.

<table border="1">
	<tbody>
		<tr>
			<th>
				Materials</th>
			<th>
				Supplier</th>
			<th>
				Catalogue number</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>CPA</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>IVF medium</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>0.25 ml plastic straw</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Sperm freezing canister</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Cane and goblet</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>Should be embryo tested</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Falcon</td>
			<td>351008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td>ABTEC</td>
			<td>TISH-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well multidish</td>
			<td>Nunc</td>
			<td>73521-424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C + 5% CO2 incubator</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LN2 storage system</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C water bath</td>
			<td>VWR</td>
			<td>89032-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>Intervet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>EMDChemicals</td>
			<td>367222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 26G 3/8 needle</td>
			<td>BD</td>
			<td>BD309625</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge &frac12; needle</td>
			<td>BD</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5135, RS-5110, RS-5005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mouse sperm cryopreservation and recovery using the i&middot;cryo kit

Miles de nuevos genéticamente modificados (GM) de las cepas de ratones han sido creados desde el advenimiento de las tecnologías de la transgénesis y el golpe de gracia. Muchos de estos valiosos animales sólo existen como animales vivos, sin un plan de copia de seguridad en caso de emergencia. La criopreservación de embriones puede ofrecer esta copia de seguridad, pero es costoso, puede ser un procedimiento prolongado y generalmente requiere de un gran número de animales para el éxito. Desde el descubrimiento de que los espermatozoides de ratón pueden ser criopreservados con éxito con un agente crioprotector básicos (CPA) que consiste de 18% de rafinosa y 3% de leche desnatada, la criopreservación de espermatozoides se ha convertido en un procedimiento aceptable y rentable para el almacenamiento, distribución y recuperación de estas cepas valiosas . Aquí se demuestra un nuevo desarrollo de I • Kit de Cryo para la criopreservación de espermatozoides de ratón. El esperma de cinco cepas de uso común de los ratones consanguíneos fueron congelados con este kit y luego se recuperó. Mayores relaciones de protección de la motilidad espermática (&gt;60%) y la motilidad progresiva rápida (&gt; 45%) en comparación con el control (básica CPA) se observó que los espermatozoides congelados con este kit de cinco cepas de ratones endogámicos. Dos etapas de desarrollo de las células de embriones después de la fecundación in vitro con el esperma recuperado se ha mejorado consistentemente en todas las cinco cepas de ratones examinados. Durante un período de 1,5 años, 49 líneas de ratones transgénicos fueron archivados por la crioconservación de esperma con el I • Kit de Cryo y más tarde recuperado por fecundación in vitro.

Watch this Scientific Journal Video about Mouse Sperm Cryopreservation and Recovery using the IÂ·Cryo Kit at JoVE.com

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Aquí se demuestra que el nuevo desarrollo • Kit de Cryo para la criopreservación de espermatozoides de ratón. Dos células etapa de desarrollo del embrión con esperma congelado-descongelado se ha mejorado consistentemente en cinco cepas de ratón con el uso de este kit. Durante un período de 1,5 años, 49 líneas de ratones genéticamente modificados fueron archivados por la crioconservación de esperma con el I • Kit de Cryo y más tarde recuperado con éxito por fecundación in vitro.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

mouse-sperm-cryopreservation-and-recovery-using-the-icryo-kit

Research

JoVE Journal

Biology

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

39.3K vistas.  Charles River. Aquí se demuestra que el nuevo desarrollo • Kit de Cryo para la criopreservación de espermatozoides de ratón. Dos células etapa de desarrollo del embrión con esperma congelado-descongelado se ha mejorado consistentemente en cinco cepas de ratón con el uso de este kit. Durante un período de 1,5 años, 49 líneas de ratones genéticamente modificados fueron archivados por la crioconservación de esperma con el I • Kit de Cryo y más tarde recuperado con éxito po...

Video: Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purificación del ADN plásmido de las colonias de bacterias Utilizando el kit de Qiagen Miniprep

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Investigación

Biología

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Inmunohistoquímica: las secciones de parafina utilizando la Vectastain ABC Kit de Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

La extracción de RNA a partir de células Neuroprecursor utilizando el kit de Bio-Rad ARN total

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genotipificación rápida de tejido de ratón utilizando extracto-N-Amp tejido Sigma PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Recolección y criopreservación de ovocitos de hámster y de embriones de ratón

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Un método de alto rendimiento para la crioconservación de esperma de pez cebra y In Vitro Fertilización

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

La criopreservación de embriones de ratón por el etileno a base de glicol vitrificación

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Evaluación del esperma de pescado utilizando Software y equipos de refrigeración

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Recogida de esperma de calidad diferencial mediante centrifugación de gradiente de densidad

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Transformación natural, expresión de proteínas y crioconservación de la filamentosa Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...